CN107475239B - Immobilization method of horseradish peroxidase and application thereof - Google Patents
Immobilization method of horseradish peroxidase and application thereof Download PDFInfo
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- CN107475239B CN107475239B CN201710741575.2A CN201710741575A CN107475239B CN 107475239 B CN107475239 B CN 107475239B CN 201710741575 A CN201710741575 A CN 201710741575A CN 107475239 B CN107475239 B CN 107475239B
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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Abstract
The invention belongs to the field of biocatalysis, and particularly relates to an immobilization method of horseradish peroxidase and application thereof. The carrier used for immobilizing the horseradish peroxidase is aminated mesoporous silica nanoparticles, and the horseradish peroxidase is immobilized by a physical adsorption method. After amination, the adsorption capacity of the mesoporous silica nanoparticles to peroxidase is improved, and the stability of the mesoporous silica nanoparticles to the peroxidase is improved. The immobilized horseradish peroxidase is applied to the treatment of phenol compounds in sewage.
Description
Technical Field
The invention belongs to the field of biocatalysis, and particularly relates to an immobilization method of peroxidase and application thereof.
Background
The enzyme is used as a biocatalyst, has the characteristics of high catalytic efficiency, high selectivity, mild reaction conditions and the like, and can be applied to various industries. However, natural enzymes are easy to inactivate, have poor stability, low recovery rate and the like, and limit various applications of the natural enzymes.
The immobilized enzyme technology is a technology which limits free enzyme in a certain area to perform active and specific catalysis by physical or chemical means and can be recycled. Compared with free enzyme, the immobilized enzyme has the following advantages: (1) the product is easy to separate from the substrate and can be repeatedly used; (2) the enzyme dosage in the enzymolysis reaction is reduced, and the cost is reduced; (3) can carry out continuous production, improves the production efficiency and is suitable for industrial application. Preparation principle of immobilized enzyme: 1) the catalytic activity and specificity of the enzyme are maintained; 2) the immobilized enzyme should have a certain stability; 3) the enzyme and the carrier have certain combination degree; 4) a suitable carrier is employed. The carrier of the immobilized enzyme is of the following general types: 1) chitosan and modification thereof; 2) cellulose and its derivatives; 3) an organic synthetic polymer; 4) a gel material; 5) magnetic particles.
With the increasing environmental pollution, people need to develop a technology which is efficient, rapid, continuous and efficient in pollutant treatment and free of secondary pollution. With the development of immobilized enzyme technology, the immobilized enzyme technology shows great superiority in environmental treatment. Common enzymes used in sewage treatment include horseradish peroxidase, laccase and the like. The immobilized horseradish peroxidase has good catalytic effect on phenolic compounds in sewage treatment. In addition, the horseradish peroxidase has the characteristics of simple preparation, low price, high specific activity, capability of adapting to a wider range of pollutant concentration and the like.
The enzyme can be immobilized by various carriers, and the mesoporous material has the advantages of uniform and adjustable pore diameter, large specific surface area, regular pore channels, stable skeleton, easy surface functionalization and the like, so that the mesoporous material is widely used for immobilization of enzyme protein at present and serves as a biocatalyst carrier. The mesoporous silica nano-particles have a certain cavity and nano-scale pore diameters which are uniformly distributed, and are beneficial to loading and immobilization of enzyme molecules; in addition, the enzyme has chemical inertia and does not influence the catalytic reaction of the enzyme.
Based on the background, the invention designs that the horseradish peroxidase is fixed by the aminated mesoporous silica nanoparticles and is used for catalytic degradation of phenol compounds in sewage, and no report is available at present.
Disclosure of Invention
Aiming at the defects of volatile activity, poor stability, low recovery rate and the like of free enzyme in catalytic reaction, the invention provides the method for removing phenolic compounds in sewage by using aminated mesoporous silica nanoparticles as immobilized carriers, which has the characteristics of large loading capacity, enzyme catalytic activity protection, easiness in recovery and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of horseradish peroxidase is characterized in that an immobilization carrier is aminated and modified mesoporous silica nanoparticles, and an immobilization method is a physical adsorption method, namely horseradish peroxidase enters the inside of the pore channels of the aminated and modified mesoporous silica nanoparticles through the physical adsorption method.
Wherein the particle size of the mesoporous silica nano-particles is 250-350nm, and the mesoporous aperture is 8-9.5 nm.
The particle size of the mesoporous silica nano-particles after amination modification is 250-350nm, and compared with that before amination, the particle size is not changed greatly; the mesoporous diameter is 7-8nm, and is reduced compared with the mesoporous diameter before amination.
Before amination, the adsorption capacity of the mesoporous silica nanoparticles to horseradish peroxidase is 24-26mg/g, and the adsorption capacity of the mesoporous silica nanoparticles to horseradish peroxidase after amination is 35-37 mg/g.
The preparation method of the immobilized horseradish peroxidase specifically comprises the following steps:
(1) uniformly mixing 3.36mL of ammonia water and 75mL of ethanol, reacting for 5min, adding 0.21mL of formaldehyde and 0.15g of resorcinol, reacting for 7h, dropwise adding 0.65mL of tetraethyl orthosilicate, after the reaction is finished, centrifuging and cleaning the obtained material with ethanol for three times, drying in vacuum for the whole night, and calcining the material at 550 ℃ for 6h to obtain mesoporous silica nanoparticles;
(2) the obtained mesoporous silica nano-particles are white powder. Dissolving the obtained white powder in ultrapure water, heating to boil, reacting for 2h, centrifuging, and drying in a vacuum drying oven; taking 0.2g of activated mesoporous silica nanoparticles, adding 2mL of 3-aminopropyltriethoxysilane, placing in 20mL of toluene, and carrying out condensation reflux at 80 ℃ for 12 h; after the reaction is finished, centrifuging the reaction solution by using absolute ethyl alcohol, and cleaning the reaction solution for three times to obtain aminated mesoporous silica nanoparticles;
(3) adding 2mg of aminated mesoporous silica nanoparticles and 10mL of horseradish peroxidase into 10mM PBS buffer solution with the pH value of 7.4, and stirring at room temperature for 24h to obtain the immobilized horseradish peroxidase.
The invention has the beneficial effects that: the invention takes the aminated mesoporous silica nanoparticles as a carrier, coats a certain amount of horseradish peroxidase, and immobilizes the horseradish peroxidase, compared with free enzyme, the stability and the recoverability of the horseradish peroxidase are improved, the catalytic activity of the horseradish peroxidase is not influenced, and the application of the horseradish peroxidase in sewage treatment is improved.
Drawings
Fig. 1 is a particle size diagram of mesoporous silica nanoparticles before and after amination.
FIG. 2 shows the adsorption amount of mesoporous silica nanoparticles to horseradish peroxidase before and after amination.
FIG. 3 is a graph showing the effect of pH on free and immobilized enzymes.
FIG. 4 shows the effect of the ratio of hydrogen peroxide to 2, 4-dichlorophenol on immobilized enzyme.
FIG. 5 is a recoverability test of an immobilized enzyme.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
Firstly, mesoporous silica nano particles are synthesized according to the following method: uniformly mixing 3.36mL of ammonia water and 75mL of ethanol, reacting for 5min, adding 0.21mL of formaldehyde and 0.15g of resorcinol, reacting for 7h, dropwise adding 0.65mL of tetraethyl orthosilicate, after the reaction is finished, centrifuging and cleaning the obtained material with ethanol for three times, drying the material in vacuum for the whole night, and calcining the material at 550 ℃ for 6h to obtain the mesoporous silica nanoparticles. The obtained mesoporous silica nano-particles are white powder. 0.2g of the resulting white powder was dissolved in ultrapure water and heated to boiling, reacted for 2 hours, centrifuged, and placed in a vacuum drying oven to be dried. And (3) adding 2mL of 3-Aminopropyltriethoxysilane (APTES) into activated mesoporous silica nanoparticles, placing in 20mL of toluene, and condensing and refluxing at 80 ℃ for 12 h. And after the reaction is finished, centrifuging the reaction solution by using absolute ethyl alcohol, and cleaning the reaction solution for three times to obtain the aminated mesoporous silica nanoparticles. The particle size of the mesoporous silica nanoparticles before and after amination is tested, and as shown in fig. 1, the particle size of the mesoporous silica nanoparticles before and after amination is not changed too much.
Horseradish peroxidase is dissolved in PBS (pH 7.4), a certain gradient is set, the concentration of the horseradish peroxidase in the PBS is 0.1 mg/mL-0.5 mg/mL, and 2mg of mesoporous silica nanoparticles and aminated mesoporous silica nanoparticles are respectively placed in PBS buffers with different horseradish peroxidase concentrations (0.1 mg/mL-0.5 mg/mL). After incubation for 24h at room temperature, the reaction solution was centrifuged, and the amount of horseradish peroxidase adsorbed to the carrier was determined by the Coomassie brilliant blue method, and the corresponding line graph was plotted, as shown in FIG. 2.
Application example 1
The concentration of 2, 4-dichlorophenol was set in the range of 0-200mg/mL, and 30mg of free enzyme and 30mg of horseradish peroxidase immobilized on aminated mesoporous silica nanoparticles were added to the 2, 4-dichlorophenol solution, respectively. The reaction started when 0-300mg/mL hydrogen peroxide was inhaled into the mixture. The optimum pH of the free enzyme and the immobilized horseradish peroxidase at pH 3.0-9.0 is shown in FIG. 3. During the immobilization, pH is one of the important influencing factors of the enzyme activity. The immobilized enzyme is more stable than the free enzyme over the pH range tested. The optimum pH for the free enzyme was 6.0, while for the immobilized enzyme was 7.0.
Application example 2
The concentration of 2, 4-dichlorophenol is set within the range of 0-200mg/ml, the enzyme immobilized on the aminated mesoporous silica nanoparticles is added to the 2, 4-dichlorophenol solution, and the ratio of hydrogen peroxide to 2, 4-dichlorophenol in the solution is changed. When the ratio of hydrogen peroxide to 2, 4-dichlorophenol is 1, the immobilized horseradish peroxidase has a high clearance rate for 2, 4-dichlorophenol, as shown in fig. 4.
Application example 3
Unlike free enzyme, immobilized horseradish peroxidase can be separated from the reaction solution and reused. The residual catalytic activity of the immobilized enzyme is expressed in recoverability. As shown in fig. 5, the activity of immobilized HRP decreased with increasing number of cycles. After seven cycles, the enzyme activity decreased to 20% of its initial activity. After five cycles of repeated experiments, more than half of the enzyme activity was lost and the phenol removal efficiency was reduced to a lower level.
Claims (5)
1. A preparation method of immobilized horseradish peroxidase is characterized by comprising the following steps: allowing horseradish peroxidase to enter the inside of the pore canal of the aminated mesoporous silica nanoparticle by a physical adsorption immobilization method; the particle size of the aminated and modified mesoporous silica nano-particles is 250-350nm, and the pore diameter of the mesoporous silica nano-particles is 7-8 nm; the preparation method comprises the following steps:
(1) uniformly mixing 3.36mL of ammonia water and 75mL of ethanol, reacting for 5min, adding 0.21mL of formaldehyde and 0.15g of resorcinol, reacting for 7h, dropwise adding 0.65mL of tetraethyl orthosilicate, after the reaction is finished, centrifuging and cleaning the obtained material with ethanol for three times, drying in vacuum for the whole night, and calcining the material to obtain mesoporous silica nanoparticles;
(2) dissolving the obtained white powder in ultrapure water, heating to boil, reacting for 2h, centrifuging, and drying in a vacuum drying oven; taking 0.2g of activated mesoporous silica nanoparticles, adding 2mL of 3-aminopropyltriethoxysilane, placing in 20mL of toluene, and condensing and refluxing; after the reaction is finished, centrifuging the reaction solution by using absolute ethyl alcohol, and cleaning the reaction solution for three times to obtain aminated mesoporous silica nanoparticles;
(3) adding 2mg of aminated mesoporous silica nanoparticles and 10mL of horseradish peroxidase into 10mM PBS buffer solution, and stirring at room temperature for 24h to obtain immobilized horseradish peroxidase;
the calcining temperature in the step (1) is 550 ℃, and the calcining time is 6 hours.
2. The method for preparing immobilized horseradish peroxidase according to claim 1, which is characterized in that: in the step (2), the condensing reflux temperature is 80 ℃, and the time is 12 hours.
3. The method for preparing immobilized horseradish peroxidase according to claim 1, which is characterized in that: the pH of the PBS buffer was 7.4.
4. An immobilized horseradish peroxidase prepared according to the method of claim 1.
5. Use of an immobilized horseradish peroxidase prepared according to the method of claim 1, wherein: the method is applied to removing the phenol compounds.
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| CN109536478A (en) * | 2017-09-21 | 2019-03-29 | 北京理工大学 | The material immobilized peroxidase of mesoporous nano, its oxidation reaction method as catalyst |
| CN108946958A (en) * | 2018-07-17 | 2018-12-07 | 中节能工程技术研究院有限公司 | A kind of enzyme mebrane reactor and the method using its processing waste water |
| CN110760500B (en) * | 2019-05-07 | 2023-03-21 | 宁波大学 | Cocrosslinking immobilization method of horseradish peroxidase |
| CN111484990B (en) * | 2020-04-21 | 2023-04-07 | 陕西师范大学 | Cobaltose peroxidase-loaded nanoreactor modified by polydopamine and prepared from cobalt hierarchical porous material and application of nanoreactor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4121946A (en) * | 1976-08-13 | 1978-10-24 | Alexei Alexeevich Chuiko | Method of producing aminated silica compounds |
| CN102344918A (en) * | 2010-08-05 | 2012-02-08 | 东北农业大学 | Enzyme stability improvement method by nano adsorption |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4121946A (en) * | 1976-08-13 | 1978-10-24 | Alexei Alexeevich Chuiko | Method of producing aminated silica compounds |
| CN102344918A (en) * | 2010-08-05 | 2012-02-08 | 东北农业大学 | Enzyme stability improvement method by nano adsorption |
Non-Patent Citations (1)
| Title |
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| 磁性介孔二氧化硅微球的制备及应用;郑杨;《中国优秀硕士论文全文数据库》;20150315;B015-123 * |
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